Aspergillus fumigatus is the leading cause of invasive mold infection and is a serious problem in immunocompromised populations worldwide. We have previously shown that survival of A. fumigatus in serum may be related to secretion of siderophores. In this study, we identified and characterized the sidA gene of A. fumigatus, which encodes L-ornithine N 5 -oxygenase, the first committed step in hydroxamate siderophore biosynthesis. A. fumigatus sidA codes for a protein of 501 amino acids with significant homology to other fungal L-ornithine N 5 -oxygenases. A stable ⌬sidA strain was created by deletion of A. fumigatus sidA. This strain was unable to synthesize the siderophores N,N؆,Nٟ-triacetylfusarinine C (TAF) and ferricrocin. Growth of the ⌬sidA strain was the same as that of the wild type in rich media; however, the ⌬sidA strain was unable to grow in low-iron defined media or media containing 10% human serum unless supplemented with TAF or ferricrocin. No significant differences in ferric reduction activities were observed between the parental strain and the ⌬sidA strain, indicating that blocking siderophore secretion did not result in upregulation of this pathway. Unlike the parental strain, the ⌬sidA strain was unable to remove iron from human transferrin. A rescued strain (⌬sidA ؉ sidA) was constructed; it produced siderophores and had the same growth as the wild type on iron-limited media. Unlike the wild-type and rescued strains, the ⌬sidA strain was avirulent in a mouse model of invasive aspergillosis, indicating that sidA is necessary for A. fumigatus virulence.

Aspergillus fumigatus is a filamentous fungus which can cause invasive disease in immunocompromised individuals. A. fumigatus can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which A. fumigatus obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM), A. fumigatus growth was supported by the addition of holotransferrin (holoTf) or FeCl 3 but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by A. fumigatus occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by A. fumigatus incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by A. fumigatus. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by A. fumigatus after 8 h of growth in MEM-serum and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by A. fumigatus even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from A. fumigatus culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that A. fumigatus survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening systemic mycosis in immunocompromised patients. We have studied the growth of A. fumigatus inside cultured cells, and the extracellular growth requirements (in serum). We measured the uptake of bound conidia by the cultured human type II pneumocyte cell line (A549) and a mouse macrophage cell line (J774). The extent of internalization was determined using a nystatin protection assay and by confocal microscopy. Both assays showed that A549 cells internalized 30% of bound conidia after three hours. In contrast, the value for J774 cells was 90%. In both J774 and A549 cells, conidia entered the endosomal pathway and ultimately co-localized with lysosomal markers. Lysosomes containing conidia were acidified. Internalized conidia germinated, and after 24-36 h of incubation with A549 cells, the hyphal tips of some intracellular germlings became exposed to the extracellular space. The importance of iron acquisition to extracellular growth was assessed by creating a strain of A. fumigatus in which the gene encoding the first step of hydroxamate siderophore biosynthesis, ornithine N5-oxygenase (AfusidA), was inactivated by gene replacement. Mutant strains were avirulent in a mouse model of invasive aspergillosis indicating that siderophore biosynthesis is a virulence factor in A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N'',N'''-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.